Induction of a physiological dispersion response in bacterial cells in a biofilm

ABSTRACT

One aspect of the present invention is directed to a composition. The composition includes a dispersion inducer comprising:
 
H 3 C—(CH 2 ) n —CH m   CH m R,
 
where   is a single or double carbon-carbon bond, m is 1 or 2, n is 2 to 15, and R is a carboxylic acid, a salt, an ester, or an amide, where the ester or amide is an isostere or biostere of the carboxylic acid. The composition additionally contains an additive component selected from one or more of the group consisting of biocides, surfactants, antibiotics, antiseptics, detergents, chelating agents, virulence factor inhibitors, gels, polymers, pastes, edible products, and chewable products. The composition is formulated so that when it is contacted with a biofilm produced by a microorganism, where the biofilm comprises a matrix and microorganism on a surface, the dispersion inducer selectively acts on the microorganism and has a suitable biological response without a required direct effect on the matrix to disperse the biofilm. The present invention is also directed to methods of using this compound.

This application claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 60/917,791, filed May 14, 2007, and 61/018,639,filed Jan. 2, 2008, which are hereby incorporated by reference in itsentirety

This invention was made with government support under grant numbers NSFMCB-0321672 and NIH R15 AI055521-01 awarded by NIH and NSF. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention is directed to a method of inducing aphysiological dispersion response in bacterial cells in a biofilm.

BACKGROUND OF THE INVENTION

Due to the compact nature of biofilm structures, the presumed reducedphysiological state of biofilm bacteria and the protection conferred bybiofilm matrix polymers, natural and artificial chemical agents areunable to adequately attack and destroy infectious biofilm populations(Costerton et al., “Bacterial Biofilms in Nature and Disease,” Annu.Rev. Microbiol. 41:435-464 (1987); Hoiby et al., “The Immune Response toBacterial Biofilms,” In Microbial Biofilms, Lappin-Scott et al., eds.,Cambridge: Cambridge University Press (1995)). Increased antibioticresistance is a general trait associated with biofilm bacteria. Whenattached, bacteria exhibit a profound resistance, rendering biofilmcells 10-1000 fold less susceptible to various antimicrobial agents thanthe same bacterium grown in planktonic (free floating) culture. Forinstance, chlorine (as sodium hypochlorite) an oxidizing biocideconsidered to be one of the most effective antibacterial agents, hasbeen shown to require a 600 fold increase in concentration to killbiofilm cells of Staphylococcus aureus when compared to planktonic cellsof the same species (Luppens et al., “Development of a Standard Test toAssess the Resistance of Staphylococcus aureus Biofilm Cells toDisinfectants,” Appl Environ Microbiol. 68:4194-200 (2002)). Severalhypotheses have been advanced to account for the extraordinaryresistance of biofilm bacteria to antibiotics including: (i) reducedmetabolic and divisional rates exhibited by biofilm bacteria(particularly those deep within the biofilm); (ii) the biofilm EPSmatrix may act as an adsorbent or reactant, reducing the amount of agentavailable to interact with biofilm cells. Additionally, the biofilmstructure may physically reduce the penetration of antimicrobial agentsby walling off access to regions of the biofilm; (iii) biofilm cells arephysiologically distinct from planktonic bacteria, expressing specificprotective factors such as multidrug efflux pumps and stress responseregulons (Brown et al., “Resistance of Bacterial Biofilms toAntibiotics: A Growth-Rate Related Effect?” J. Antimicrob. Chemotherapy22:777-783 (1988); Anwar et al., “Establishment of Aging Biofilms:Possible Mechanism of Bacterial Resistance to Antimicrobial Therapy,”Antimicrob. Agents Chemother. 36:1347-1351 (1992); Mah et al.,“Mechanisms of Biofilm Resistance to Antimicrobial Agents,” TrendsMicrobiol. 9:34-39 (2001); Sauer et al., “Pseudomonas aeruginosaDisplays Multiple Phenotypes During Development as a Biofilm,” J.Bacteriol. 184:1140-1154 (2002); Stewart, P. S., “Mechanisms ofAntibiotic Resistance in Bacterial Biofilms,” Int. J. Med. Microbiol.292:107-113 (2002); Donlan et al., “Biofilms: Survival Mechanisms ofClinically Relevant Microorganisms,” Clinical Microbiol. Reviews15:167-193 (2002); Gilbert et al., “The Physiology and CollectiveRecalcitrance of Microbial Biofilm Communities,” Adv. Microb. Physiol.46:202-256 (2002); Gilbert et al., “Biofilms In vitro and In vivo: DoSingular Mechanisms Imply Cross-Resistance?” J. Appl. Microbiol. Suppl.98S-110S (2002)). As detailed molecular studies emerge, it is becomingapparent that each of these factors plays a role in the unusualresistance of biofilms to antimicrobials. Initial treatment is usuallyeffective in killing bacteria only at the margins of biofilmmicrocolonies. Bacteria deep within these microcolonies are unaffectedby the antibiotic and form a nidus for continued dissemination of theinfection.

Microbial biofilms in infections and in industrial systems presentsignificant problems due to their recalcitrance to effective treatment.

Detachment is a generalized term used to describe the removal of cells(either individually or in groups) from a biofilm or substratum. Bryers,J. D., “Modeling Biofilm Accumulation,” In: Physiology Models inMicrobiology. Bazin et al., eds., Boca Raton, Fla., Vol. 2, pp. 109-144(1988) categorized four distinct detachment mechanisms by which bacteriadetach from a biofilm. These are: abrasion, grazing, erosion, andsloughing. These mechanisms have been described principally from thepoint of view of the chemical and physical environment acting uponbiofilm bacteria. Active detachment as a physiologically regulated eventhas been hinted at by many authors, but few studies have been performedto demonstrate a biological basis for detachment of bacteria from abiofilm.

One study on the physiological regulation of detachment was carried outby Peyton et al., “Microbial Biofilms and Biofilm Reactors,” BioprocessTechnol. 20:187-231 (1995) on P. aeruginosa. In their work, it wasobserved that substrate limitation resulted in a decrease in thedetachment rate, presumably a result of reducing the growth rate.Allison et al., “Extracellular Products as Mediators of the Formationand Detachment of Pseudomonas fluorescens Biofilms,” FEMS Microbiol.Lett. 167:179-184 (1998) showed that following extended incubation, P.fluorescens biofilms experienced detachment, coincident with a reductionin EPS. In Clostridium thermocellum, the onset of stationary phase hasbeen correlated with increased detachment from the substratum (Lamed etal., “Contact and Cellulolysis in Clostridium thermocellum via ExtensiveSurface Organelles,” Experientia 42:72-73 (1986)). It has beenpostulated that starvation may lead to detachment by an unknownmechanism which allows bacteria to search for habitats richer innutrients (O'Toole et al., “Biofilm Formation as Microbial Development,”Ann. Rev. Microbiol. 54:49-79 (2000)).

The transition from a flowing system to a batch culture system has beenobserved by many labs to result in biofilm detachment. One lab hasobserved reproducible detachment of biofilm cells of P. aeruginosa whenflow is arrested in a continuous culture system (Davies, D. G.,“Regulation of Matrix Polymer in Biofilm Formation and Dispersion,” InMicrobial Extracellular Polymeric Substances, pp. 93-112, Wingender etal., eds., Berlin: Springer (1999)).

The release of degradative enzymes has been proposed by others. One suchexample is found with the gram positive organism Streptococcus mutanswhich 30 produces a surface protein releasing enzyme (SPRE), shown tomediate the release of proteins from the cell envelope (Lee et al.,“Detachment of Streptococcus mutans Biofilm Cells by an EndogenousEnzymatic Activity,” Infect. Immun. 64:1035-1038 (1996)). Boyd et al.,“Role of Alginate Lyase in Cell Detachment of Pseudomonas aeruginosa,”Appl. Environ. Microbiol. 60:2355-2359 (1995) showed thatover-expression of alginate lyase causes the degradation of alginate.When a mucoid strain of P. aeruginosa was induced to over-expressalginate lyase, cells were more easily removed by gentle rinsing fromsolid medium.

Cell density dependent regulation may also be responsible for therelease of enzymes which can degrade biofilm matrix polymers allowingbacteria to disperse from a biofilm. It has been observed at the Centerfor Biofilm Engineering at Montana State University, USA (Davies, D. G.and Costerton, J. W.) and in the laboratories of Dr. Lapin-Scott at theUniversity of Exeter, UK, that when certain bacteria (including P.aeruginosa) reach high cell densities in biofilm cell clusters, thebacteria often undergo a detachment event. Mutants of P. aeruginosawhich lacked the ability to synthesize the quorum sensing autoinducer3OC₁₂-HSL, were susceptible to detachment following treatment with milddetergent (Davies et al., “The Involvement of Cell-to-Cell Signals inthe Development of a Bacterial Biofilm,” Science 280:295-298 (1998)).Other investigators have demonstrated that homoserine lactones may playa role in detachment. Lynch et al., “Investigation of Quorum Sensing inAeromonas hydrophila Biofilms Formed on Stainless Steel,: In:Biofilms—The Good, the Bad and the Ugly, Wimpenny et al., eds. Bioline,Cardiff. pp. 209-223 (1999) reported an increase in detachment ofAeromonas hydrophila from biofilms and Puckas et al., “A Quorum Sensingsystem in the Free-Living Photosynthetic Bacterium Rhodobactersphaeroides,” J. Bacteriol. 179:7530-7537 (1997) reported thathomoserine lactone production was negatively correlated with cellcluster formation in Rhodobacter sphaeroides.

It has been recognized that P. aeruginosa biofilms do not develop intomacroscopic biofilm structures in batch culture flasks (at the glassliquid interface). Yet, when medium is pumped continuously through sucha flask, (as in a chemostat) a luxurious biofilm develops completelycoating the glass surface. When flow is halted in such a system, thebiofilm sloughs after a number of days, generally around 72 hrs (Davieset al., “The Involvement of Cell-to-Cell Signals in the Development of aBacterial Biofilm,” Science 280:295-298 (1998)). The inability ofbiofilms to develop in batch culture has been observed for a number ofgram negative and gram positive bacteria as well as mixed cultures ofbacteria. This phenomenon demonstrates that there is some aspect ofbatch growth that is inhibitory to biofilm development.

During the last stage of biofilm development, the protein profile ofbacteria matches more closely the protein profile of planktonic cellsthan it does biofilm bacteria from the previous stage, denotedmaturation II (see FIG. 3 of the current application, and Sauer et al.,“Pseudomonas aeruginosa Displays Multiple Phenotypes During Developmentas a Biofilm,” J. Bacteriol. 184:1140-1154 (2002)).

Due to the compact nature of biofilm structures, the presumed reducedphysiological state of biofilm bacteria and the protection conferred bybiofilm matrix polymers, current natural and artificial chemical agentsare unable to adequately attack and destroy infectious biofilmpopulations (Costerton et al., “Bacterial Biofilms in Nature andDisease,” Annu. Rev. Microbiol. 41:435-464 (1987); Hoiby et al., “TheImmune Response to Bacterial Biofilms,” In Microbial Biofilms,Lappin-Scott et al., eds., Cambridge: Cambridge University Press(1995)).

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a composition. Thecomposition comprises one or more dispersion inducers having thefollowing formula:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 2 to 15, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid. Thecomposition additionally contains one or more additive componentsselected from the group consisting of biocides, surfactants,antibiotics, antiseptics, detergents, chelating agents, virulence factorinhibitors, gels, polymers, pastes, edible products, and chewableproducts. The composition is formulated so that when it is contactedwith a biofilm produced by a microorganism, where the biofilm comprisesa matrix and microorganism on a surface, the dispersion inducerselectively acts on the microorganism and has a suitable biologicalresponse without a required direct effect on the matrix to disperse thebiofilm.

Another aspect of the present invention relates to a method of treatingor preventing a condition mediated by a biofilm in a subject. The methodcomprises providing a subject having, or susceptible to, a conditionmediated by a biofilm produced by a microorganism, whereby the biofilmcomprises a matrix and the micro-organism on a surface. Administered tothe subject is a dispersion inducer comprising:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 2 to 15, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid, underconditions effective for the dispersion inducer to selectively act onthe microorganism and have a suitable biological response without arequired direct effect on the matrix. As a result, the conditionmediated by a biofilm in the subject is treated or prevented.

An additional aspect of the present invention relates to a method oftreating or inhibiting formation of a biofilm on a surface. This methodinvolves providing a surface having or being susceptible to formation ofa biofilm produced by a microorganism, where the biofilm comprises amatrix and the micro-organism on the surface. Administered to thesurface is a dispersion inducer comprising:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 2 to 15, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid, underconditions effective for the dispersion inducer to selectively act onthe microorganism and have a suitable biological response without arequired direct effect on the matrix. As a result, formation of thebiofilm on the surface is treated or inhibited.

Another aspect of the present application relates to a solutioncomprising:

a dispersion inducer having the following formula:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 4 to 7, andR is a carboxylic acid, where said inducer is present at a concentrationless than 0.5 percent by weight, and where said solution has a pHgreater than 5.

A further aspect of the present invention is directed to a compositioncomprising: a component selected from one or more of the groupconsisting of biocides, surfactants, antibiotics, antiseptics,detergents, chelating agents, virulence factor inhibitors, gels,polymers, pastes, edible products, and chewable products. In addition,the composition includes a dispersion inducer comprising:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 4 to 7, andR is a carboxylic acid. The inducer is formulated in a non-salt form.

The present invention is also directed to a solution which includes acis isomer of 2-decenoic acid, where the solution is selected from thegroup consisting of a skin cream, a toothpaste, and a mouthwash andwhere the solution is substantially free of the trans isomer of2-decenoic acid.

Another form of the present application is directed to a solutioncomprising: a cis isomer of 2-decenoic acid, where the solution isselected from the group consisting of a skin cream, a toothpaste, and amouthwash and where said solution is trans isomer-free.

Another form of the present application is for a method which comprisesproviding contact lenses and a solution comprising a dispersion inducerat a concentration less than 0.5% by weight, said inducer comprising:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 4 to 7, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid. The contactlenses are then treated with said solution.

A further form of the present invention is for a method which involvesproviding a subject with a skin condition and a solution having a pHgreater than 5, where the solution comprising a dispersion inducer at aconcentration less than 0.5% by weight said inducer comprising:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 4 to 7, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid. The skincondition is then treated with the solution.

Further aspects of the present invention relate to methods of: treatingsubjects with burns; treating and/or preventing dental plaque, dentalcaries, gingival disease, and oral infection; cleaning and/ordisinfecting contact lenses; treating and/or preventing acne or otherbiofilm-associated skin infections on the skin of a subject, andtreating and/or preventing a chronic biofilm-associated disease in asubject. The methods involve administering the dispersion induceraccording to the present invention, under conditions effective toaccomplish each respective task. Advantageously, the biofilm dispersioninducer is highly bioactive on the microorganisms within the biofilm,and, therefore, the pharmaceutically acceptable formulation need not bechemically or mechanically active to disrupt the matrix directly. Thus,the composition may have a mild pH and be non-irritating.

The present invention also relates to a composition comprising one ormore dispersion inducers and one or more additive components. Theseadditive components are selected from the group consisting of biocides,surfactants, antibiotics, antiseptics, detergents, chelating agents,virulence factor inhibitors, gels, polymers, pastes, edible products,and chewable products. The composition is formulated so that when it iscontacted with a biofilm produced by a microorganism, where the biofilmcomprises a matrix and microorganism on a surface, the dispersioninducer selectively acts on the microorganism and has a suitablebiological response without a required direct effect to disrupt thematrix.

Another aspect of the present invention relates to a method of treatingor preventing a condition mediated by a biofilm in a subject. Thismethod involves providing a subject having, or susceptible to, acondition mediated by a biofilm produced by a microorganism, whereby thebiofilm comprises a matrix and the micro-organism on a surface. Adispersion inducer is administered to the subject under conditionseffective for the dispersion inducer to selectively act on themicroorganism and have a suitable biological response without a requireddirect effect on the matrix, whereby the condition mediated by a biofilmin the subject is treated or prevented.

A further embodiment of the present application is directed to a methodof treating or inhibiting formation of a biofilm on a surface. Thisinvolves providing a surface having or being susceptible to formation ofa biofilm produced by a microorganism, whereby the biofilm comprises amatrix and the micro-organism on the surface. A dispersion inducer isadministered to the surface under conditions effective for thedispersion inducer to selectively act on the microorganism and have asuitable biological response without a required direct effect on thematrix, whereby formation of the biofilm on the surface is treated orinhibited.

The present invention addresses the “biofilm problem” by artificiallyinducing bacteria to undergo physiological process of biofilmdispersion. The ability to induce dispersion will allow the control ofbiofilms directly and will improve existing treatments with biocides,topical antibiotics, detergents, etc. The examples of situations inwhich artificial dispersion would be of benefit include improvedcleaning of contact lenses and teeth, improved antiseptic activity inthe home, in industry, and in the medical community and enhanced cidalactivity for existing antibiotic treatments such as with burn patientsinfected with Pseudomonas aeruginosa.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are schematic representations of biofilm treated with anantibiotic and/or a dispersion inducer. As shown in FIG. 1A, followingtreatment, only the cells on the surface of the biofilm are killed bythe antibiotic. FIG. 1B is a schematic representation of a biofilminduced to disperse along with treatment with antibiotic. Dispersedcells are completely killed during the treatment.

FIGS. 2A-C depict the effect of addition of chloroform extracted spentculture medium (CSM) which contains a dispersion inducing compound, tomature biofilms of Pseudomonas aeruginosa. FIG. 2A shows biofilm growingin continuous culture on a glass slide in a flow cell. FIG. 2B shows thesame area of biofilm 5 minutes after the addition of the dispersioninducer. FIG. 2C shows complete dis-aggregation of biofilm following 30minutes initial treatment with dispersion inducer.

FIG. 3A is a schematic representation of the life cycle of a biofilm. 1,Planktonic bacteria are transported (actively and passively) to thesubstratum. 2, Cell surface molecules interact with the substratumresulting in reversible surface attachment. 3, Phenotypic changes in thebacterial cell result in cell surface modifications and the productionof extracellular polymeric substances, which irreversibly cement thecells to the surface. 4, Physiological changes continue with alterationsin metabolism, cell-cell signaling and morphology as biofilm maturationoccurs. 5, Cells release degradative enzymes to digest matrix polymermaterial and alter surface appendages as biofilm detachment occurs. Theseries of photomicrographs at the bottom of FIG. 3B, show, in order,phase contrast photomicrographs of the five stages of biofilmdevelopment by P. aeruginosa grown in continuous culture in a flow-celland imaged by microscopy. (Sauer et al., “Pseudomonas aeruginosaDisplays Multiple Phenotypes During Development as a Biofilm,” J.Bacteriol. 184:1140-1154 (2002), which is hereby incorporated byreference in its entirety).

FIGS. 4A-B are phase contrast photomicrographs of biofilm 20 dispersioninduced by cessation of flow. FIG. 4A shows early biofilm developmentunder flowing conditions, while FIG. 4B is the same location after flowis stopped for 72 hrs.

FIG. 5 is a graph showing the effect of chloroform extraction on spentmedium dispersion activity. Biofilms were cultured for six days incontinuous culture in EPRI medium in biofilm tube reactors (Sauer etal., “Pseudomonas aeruginosa Displays Multiple Phenotypes DuringDevelopment as a Biofilm,” J. Bacteriol. 184:1140-1154 (2002), which ishereby incorporated by reference in its entirety) and treated with spentmedium (control) and chloroform extracted spent medium (CSM). Celldispersion was determined as the optical density of culture effluentcollected at the end of culture tubes. The error bar represents standarddeviation for three replicate experiments.

FIG. 6A shows microcolonies of P. aeruginosa biofilms grown incontinuous culture demonstrating native dispersion response. During thedispersion stage of biofilm development, bacteria become motile withincell clusters and exit to the bulk liquid through a breach in themicrocolony wall. Each photomicrograph shows a microcolony whoseinterior has been voided in this manner. The arrow indicates thelocation of a void. Images taken at 1000× magnification; bar represents10 μm. FIG. 6B is a transmitted light image, and FIG. 6C is afluorescent image showing the size dependence of dispersion response.Biofilm microcolonies growing in continuous culture having dimensions ofgreater than 40 μm diameter×10 μm thickness show dispersion (left 3).Microcolonies below this minimum dimension remain “solid” (right 2photomicrographs). Fluorescence indicates presence of cells (lacZreporter on chromosome). All images are the same relative size at 500×magnification; bars represent 40 μm. Arrows indicate void areas withinmicrocolony.

FIGS. 7A-D show treatment of P. aeruginosa mature biofilms with spentmedium, CSM and cis-2-decenoic acid. As shown in FIG. 7A, at 30 min,biofilms grown in silicone tubing were exposed to spent medium or freshmedium. Bacteria in effluent were collected continuously for 100 min andcell density determined by OD₆₀₀. As shown in FIG. 7B, biofilm grown incontinuous culture in silicone tubing for 4 days and switched either tofresh medium for 1 hr, or CSM for 1 hr. Extruded contents of controltube shows intact biofilm. Extruded contents of CSM-treated biofilmshows dispersion. Photomicrographs show addition of CSM to maturebiofilm grown in continuous culture in a microscope-mounted flow cell,as shown in FIG. 7C. Microcolony disaggregation is shown to begin at 7min. After 30 min exposure, the microcolony had completelydisaggregated. Dispersed cells were actively motile (not visible instatic image), indicating a change in phenotype compared to cells inintact microcolony (prior to CSM addition). As shown in FIG. 7D,addition of 10 μM cis-2-decenoic acid (cis-DA) to mature biofilm grownin continuous culture in a microscope-mounted flow cell. Microcolonydisaggregation is shown to begin at 11 min. Complete microcolonydisaggregation is shown within 30 min exposure. Control biofilms treatedwith carrier fluid were not affected by treatment up to 1 hr.

FIGS. 8A-B show biofilm development in the continuous presence of CSMdiluted in modified EPRI to concentration of Spent medium. Averagethickness (FIG. 8A) and surface area of biofilms grown in the presenceof CSM (FIG. 8B) are significantly less than for untreated biofilms.Grey bars, biofilms treated with CSM. Black bars, biofilms grown in theabsence of dispersion inducer. Error bars represent one standarddeviation for 20 randomly selected microcolonies

FIG. 9 shows dispersion of different bacterial biofilms by P. aeruginosaCSM using microtiter plate dispersion bioassay. Y-axis indicates numberof cells released into the bulk liquid of 16 replicate wells in 3replicate experiments, following treatment for 1 hr with CSM or carriercontrol (−), containing sterile medium. Hatched line indicates level ofdispersion in carrier control samples. All differences between CSMsamples and controls are statistically significant at indicated P-valueas determined by Student's T-test.

FIGS. 10A-C show a microtiter plate dispersion bioassay. FIG. 10A showsoptical densities of cells released from biofilm-containing microtiterplate wells. White bar, control sample treated with EPRI alone. Greybar, sample treated with CSM. Black bars represent biofilms treated withC-18 reverse phase HPLC fractions of CSM eluted in an acetonitrilegradient from 2% to 75%. Results are the average of 16 replicate wells,error bars represent one standard deviation. Results from Student'sT-test show P<0.001 for CSM and 22-minute HPLC samples. FIG. 10B showsmicrotiter plate biofilm dispersion bioassay comparing variousconcentrations of cis-2-decenoic acid to spent medium. Optical densitiesof cells released from biofilm-containing microtiter plate wells.Negative control wells contained P. aeruginosa treated with 10% ethanolin EPRI. Grey bar represents biofilms treated with spent medium. Blackbars represent biofilms treated with increasing concentrations ofcis-2-decenoic acid in 10% ethanol. Results are the average of 16replicate wells, error bars represent one standard deviation Student'sT-test indicated P<0.001 for all cis-2-decenoic acid samples compared tocontrol. FIG. 10C shows the structure of cis-2-decenoic acid.

FIG. 11 shows dispersion of different bacterial biofilms bycis-2-decenoic acid using microtiter plate dispersion bioassay. Y-axisindicates number of cells released into the bulk liquid of 16 replicatewells in 3 replicate experiments, following treatment for 1 hr. with0.01 μM cis-2-decenoic acid (CDA), or carrier control (−), containingmedium+10% ethanol. Hatched line indicates level of dispersion incarrier control samples. All differences between cis-2-decenoic acidtreated samples and controls are statistically significant at indicatedP-value as determined by Student's T-test.

FIGS. 12A-C show the spectral analysis of P. aeruginosa CSM andcis-2-decenoic acid. FIG. 12A shows product ion mass peaks for the 171M/Z molecule detected in active HPLC CSM fraction and for syntheticcis-2-decenoic acid. Y-axis indicates intensity; X-axis indicates M/Z inpositive ion mode. CSM sample matches peaks from syntheticcis-2-decenoic acid Note that in mass spectrometry, peak intensity isnot a direct indication of concentration. FIG. 12B shows GC-MS spectrumof P. aeruginosa CSM and cis-2-decenoic acid. CSM sample Peak at 15.9min, indicates solvent carrier. Y-axis indicates intensity; X-axisindicates time in minutes. FIG. 12C shows FT-IR spectrum of P.aeruginosa CSM and cis-2-decenoic acid. Y-axis indicates absorbance;X-axis indicates reciprocal centimeters.

FIG. 13 shows the addition of 10 μM cis-2-decenoic acid (cis-DA) tomature biofilm grown in continuous culture in a microscope-mounted flowcell. Microcolony disaggregation is shown to begin at 11 min. Completemicrocolony disaggregation is shown within 15 min exposure. Controlbiofilms treated with carrier fluid were not affected by treatment up to1 hr.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is directed to a composition. Thecomposition includes one or more dispersion inducers comprising:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 2 to 15, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid. Thecomposition additionally contains one or more additive componentsselected from the group consisting of biocides, surfactants,antibiotics, antiseptics, detergents, chelating agents, virulence factorinhibitors, gels, polymers, pastes, edible products, and chewableproducts. The composition is formulated so that when it is contactedwith a biofilm produced by a microorganism, where the biofilm comprisesa matrix and microorganism on a surface, the dispersion inducerselectively acts on the microorganism and has a suitable biologicalresponse without a required direct effect on the matrix to disperse thebiofilm. In achieving this result, the dispersion inducer of the presentinvention can act in preference directly on the matrix. Alternatively,the dispersion inducer can act on the microorganism which, in turn, actsto disrupt the matrix. This effect may also involve not relying on adirect effect on the matrix. Typically, the biofilm inducer will have noeffect on the matrix directly or be present at a concentration where nodirect effect on the matrix is evident. On the other hand, the range ofeffective concentrations suggest a biochemical response mechanism in themicroorganisms, wherein the dispersion inducer mimics an intercellularcommunication composition. The composition acts to induce a dispersionresponse by the bacteria, which in turn is responsible for release ofthe bacteria from the biofilm. Additionally, the composition is able toact on bacteria not in a biofilm (planktonic bacteria), inducing thesebacteria to mount a physiological response which prevents the formationof a biofilm. Additional components of a composition may be directed todisrupting or removing the matrix from the surface or substrate. Forexample, the composition may comprise a dentifrice adapted to abrasivelyremove plaque from teeth.

The R group of the above inducer may be selected from the groupconsisting of:

Alternatively, R can be a homoserine lactone or a furanone group. Thecomposition also includes an additive component such as one or more ofbiocides, surfactants, antibiotics, antiseptics, detergents, chelatingagents, virulence factor inhibitors, gels, polymers, pastes, edibleproducts, or chewable products.

The dispersion inducer of the present invention desirably comprises 7-10carbon atoms. It is preferred that this inducer be a carboxylic acid(e.g., a monounsaturated fatty acid). It is more preferred that thedispersion inducer comprise:CH₃—(CH₂)_(n)—CH═CHCOOHSuitable non-salt forms of this dispersion inducer being the followingrespective cis- and trans-isomers:

Of these, the cis isomer is preferred.

Other suitable alkanoic acids include hexanoic acid, heptanoic acid,octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoicacid, tridecanoic acid, tetradecanoic acid, pentanoic acid, hexadecanoicacid, heptadecanoic acid, octadecanoic acid, and nonadecanoic acid.

Useful alkenoic acids include 2-hexenoic acid, 2-heptenoic acid,2-octenoic acid, 2-nonenoic acid, 2-undecenoic acid, 2-dodecenoic acid,2-tridecenoic acid, 2-tetradecenoic acid, 2-pentadecenoic acid,2-hexadecenoic acid, 2-heptadecenoic acid, 2-octadecenoic acid, and2-nonadecenoic acid. These may be cis or trans isomers.

The composition of the present invention can be formulated at a numberof pH ranges, to treat different types of bacteria, as follows: 1.5 to4.5 for acid loving bacteria; 4.5 to 8.0 for acid tolerant bacteria; 6.8to 7.4 for substantially neutral pH loving bacteria; and 8.0 to 9.8 foralkali tolerant bacteria. An essentially neutral pH is particularlydesirable for subjects with acid reflux. The concentration of thedispersion inducer can be 0.01 μM to 30 mM.

The composition can be entirely or substantially (i.e. less than 10 wt%) ethanol free and/or formaldehyde free.

A surface (or substrate) coated with the composition is also encompassedby the present invention.

Another aspect of the present invention relates to a method of treatingor preventing a condition mediated by a biofilm in a subject. The methodcomprises providing a subject having, or susceptible to, a conditionmediated by a biofilm produced by a microorganism, whereby the biofilmcomprises a matrix and the micro-organism on a surface. Administered tothe subject is a dispersion inducer comprises:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 2 to 15, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid, underconditions effective for the dispersion inducer to selectively act onthe microorganism and have a suitable biological response without arequired direct effect on the matrix. As a result, the conditionmediated by a biofilm in the subject is treated or prevented. The methodof dispersing the biofilm may further include administering to thebiofilm, in conjunction with administering the dispersion inducer, anantimicrobial treatment. The treatment can be the administration ofbiocides (e.g., hydrogen peroxide), surfactants, antibiotics,antiseptics, detergents, chelating agents, virulence factor inhibitors,gels, polymers, pastes, edible products, chewable products, ultrasonictreatment, radiation treatment, thermal treatment, and/or mechanicaltreatment.

An additional aspect of the present invention relates to a method oftreating or inhibiting formation of a biofilm on a surface. This methodinvolves providing a surface having or being susceptible to formation ofa biofilm produced by a microorganism, where the biofilm comprises amatrix and the micro-organism on the surface. Administered to thesurface is a dispersion inducer comprising:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 2 to 15, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid, underconditions effective for the dispersion inducer to selectively act onthe microorganism and have a suitable biological response without arequired direct effect on the matrix. As a result, formation of thebiofilm on the surface is treated or inhibited.

In one embodiment, the surface to be treated includes indwelling medicaldevices, such as catheters, respirators, and ventilators. In addition,the surface can be in implanted medical devices, including stents,artificial valves, joints, pins, bone implants, sutures, staples,pacemakers, and other temporary or permanent devices. The dispersioninducer of the present invention can also be included in surgical glue.In another embodiment, the surface to be treated includes drains, tubs,kitchen appliances, countertops, shower curtains, grout, toilets,industrial food and beverage production facilities, flooring, and foodprocessing equipment. In a further embodiment, the surface to be treatedis a heat exchanger surface or a filter surface. Thus, treatmentprovides a means for reducing the degree of biofouling of the heatexchanger or filter. In a final embodiment, the surface to be treated isa marine structure which includes boats, piers, oil platforms, waterintake ports, sieves, and viewing ports. The surface can alternativelybe associated with a system for water treatment and/or distribution(e.g., a system for drinking water treatment and/or distributing, asystem for pool and spa water treatment, a system for treatment and/ordistribution of water in manufacturing operations, and a system fordental water treatment and/or distribution). The surface can also beassociated with a system for petroleum drilling, storage, separation,refining and/or distribution (e.g., a petroleum separation train, apetroleum container, petroleum distributing pipes, and petroleumdrilling equipment). The dispersion inducer can also be included informulations directed at reducing or eliminating biofilm deposits orbiofouling in porous medium, such as with oil and gas bearing geologicalformations. The treatment may be accomplished by applying a coating,such as paint, to the surface.

The method of inhibiting formation of a biofilm on a surface may furtherinvolve administering to the surface, in conjunction with administeringthe dispersion inducer, an antimicrobial treatment. The treatment can beadministration of biocides, surfactants, antibiotics, antiseptics,disinfectants, medicines, detergents, chelating agents, virulence factorinhibitors, ultrasonic treatment, radiation treatment, thermaltreatment, and mechanical treatment. In one embodiment, the dispersioninducer and the antimicrobial treatment are administered simultaneously.In another embodiment, the dispersion inducer and antimicrobialtreatment are administered separately.

The dispersion inducer can be impregnated in a surface in order toinhibit formation of a biofilm on the surface. Alternatively, thedispersion inducer can be in a copolymer or a gel coating over thesurface.

The present invention also relates to a method of treating subjects withburns. The method involves administering the dispersion induceraccording to the present invention, under conditions effective to treatburns in the subject. A specific application of the invention provides atopical dressing for burn patients comprising dispersion inducingmolecules or their natural or synthetic analogs to prevent thedevelopment of infectious biofilms or to disperse the cells of existinginfectious biofilms.

The present invention further relates to a method of treating and/orpreventing dental plaque, dental carries, gingival disease, periodontaldisease, and oral infection in a subject. The method involves treatingthe oral cavity of the subject with the dispersion inducer according tothe present invention. Treating can be carried out with a dentifrice,mouthwash, dental floss, gum, strip, toothpaste, a toothbrush containingthe dispersion inducer, and other preparations containing the dispersioninducer. The composition may also contain other compounds known in thedental arts that are typically added to dental compositions. Forexample, the dispersion inducer composition may also include fluoride,desensitizing agents, anti-tartar agents, anti-bacterial agents,remineralization agents, whitening agents, and anti-caries agents.

The amount of dispersion inducer present will vary dependent on thedental composition that contains the dispersion inducer. It has beenfound that the dispersion inducer is active over a wide range ofconcentrations against oral bacteria. For instance, the dispersioninducer may be present in an amount ranging from 0.1 nM to 10 mM.However, lower and higher concentrations may be used depending on thedental composition, the other components present in the dispersioninducer composition, and various other factors appreciated by those ofskill in the art. The known properties of the dispersion inducer, suchas its fatty acid characteristics and its hydrophobicity, will assist askilled artisan in determining how much of the dispersion inducer shouldbe used, determining how the compound will chemically interact withother components, and providing other useful information about thecompound.

Specific dental applications and dental compositions are contemplated inthis invention. In this regard, the invention relates to a toothbrushcontaining a dispersion inducer composition. Toothbrushes, as is wellknown in the art, contain a plurality of bristles and a solid support onwhich the bristles are mounted, where the solid support includes a brushhead having a plurality of tuft holes that receive the bristles.Variations and modifications of the basic toothbrush are well known inthe art. See, for example, U.S. Pat. No. 7,251,849, herein incorporatedby reference in its entirety.

The dispersion inducer of this invention has a chemical formula as setforth above. Additional components that may be included in thedispersion inducer compositions are also set forth above. The dispersioninducer composition may be incorporated in the various parts of thetoothbrush by means known in the art. For instance, the dispersioninducer composition may be contained in the tuft holes of thetoothbrush. See U.S. Pat. No. 5,141,290, herein incorporated byreference in its entirety, for an example of how a composition can becontained within the tuft holes of a toothbrush. Alternatively, thedispersion inducer composition may be coated or embedded in the bristlesof the toothbrush.

Other parts of the toothbrush may also be coating or embedded with thedispersion inducer composition, including any parts of the toothbrushthat supplement the bristles and are designed to be contacted with theoral cavity. For example, it is common for toothbrushes to containrubber paddles, tongue cleaners, or other pieces extended from the headfor the purposes of being contacted with the tooth, tongue, gums, orother areas of the oral cavity. These parts may be embedded with thedispersion inducer composition and, optionally, a surfactant, biocide,and/or other additive discussed above.

To assist in controlling the release of the dispersion inducer from thetoothbrush, the dispersion inducer composition may contain an agent thatinteracts with the dispersion inducer to assist in the controlledrelease. The agent may interact with the dispersion inducer in a mannerthat the release is either accelerated or prolonged, depending on thedesired use. The level of controlled release can also depend on howeasily or difficult the dispersion inducer adheres to the portion of thetoothbrush that it is applied to. In a preferred embodiment, thedispersion inducer is slowly released from the toothbrush over repeatedbrushings. Agents that enable the slow release of an active ingredientare well known to those of skill in the art.

The controlled release may also be effectuated by encapsulating thedispersion inducer in an encapsulated system that allows a controlledrelease. In this embodiment, the dispersion inducer composition ispreferably in the form of a plurality of small microspheres thatencapsulate the dispersion inducer. The microspheres can have an outercoating of dissolvable material that enables the dispersion inducer toslowly release over repeated brushings. Suitable microspheres includethose disclosed in U.S. Pat. No. 5,061,106, herein incorporated byreference in its entirety.

This invention also relates to a toothpaste composition that contains(a) fluoride and/or a remineralization agent; (b) an orally-acceptedvehicle; and (c) a dispersion inducer composition. The dispersioninducer of this invention has a chemical formula as set forth above.Additional components that may be included in the dispersion inducercompositions are also set forth above. Often, toothpastes also containsodium lauryl sulfate or other sulfates.

Fluoride in its various forms is a common active ingredient intoothpaste to prevent cavities and promote the formation of dentalenamel and bones. Any fluoride source, such as fluoride salts may beused in the toothpaste of this invention. Preferably, the fluoride issodium fluoride (NaF) or sodium monofluorophosphate (Na₂PO₃F).Typically, the amount of fluoride present in the toothpaste ranges from100 to 5000 parts per million fluoride ion, preferably 1000 to 1100parts per million.

In certain instances, it is preferable to replace or supplement thefluoride with a remineralization agent. Remineralization, in the contextof dental usage, generally refers to treating the teeth so as to preventdental caries, or decrease their chance of occurring, and otherwiseenhance the teeth so that they can return to their original, healthystate. While fluoride can be considered a remineralization agent, otheragents often take the place of fluoride or supplement fluoride toprovide the toothpaste with a stronger cleansing or remineralizationproperties. Common remineralization agents are calcium salts, such ascalcium phosphate, calcium sulfate, anhydrous calcium sulfate, calciumsulfate hemihydrate, calcium sulfate dihydrate, calcium malate, calciumtartrate, calcium malonate, and calcium succinate. Hydroxyapitatenanocrystals and zinc compounds have also been shown to be effectiveremineralization agents.

The orally-accepted vehicle may be any vehicle known in the art that canbe used to deliver the fluoride and/or remineralization agent, anddispersion inducer to the teeth of a patient. The orally-acceptedvehicle may also be glycerin, propylene glycol, polyethylene glycol,triglyceride, diglyceride, mineral oil, organic oils, essential oils,fatty vegetable oils, and combinations thereof. Often these vehicles areused in combination with water or a water-based solvent.

The toothpaste composition may contain other components of toothpasteswell known in the art. For instance, the toothpaste composition maycontain baking soda, enzymes, vitamins, herbs, calcium compounds such ascalcium sodium phosphosilicate, coloring agents, and/or flavoringagents. Desensitizing agents may also be added. As known in the art,desensitizing agents can reduce sensitivity in teeth by treatingsensitivities caused by demineralization or suppressing the sensitivitysymptoms by desensitizing the nerves. The composition may also containan antibacterial or an antiplaque agent. Antibacterial agents arepreferable included in the composition to prevent gingivitis,periodontitis, and other oral diseases. Suitable antibacterial agentsinclude triclosan, zinc chloride, chlorhexidine, benzthonium chloride,and cetyl pyridinium chloride.

This invention also relates to an oral composition for treating and/orpreventing dental plaque, gingival diseases, periodontal diseases,and/or oral infection. The oral composition contains an orally-acceptedvehicle and a dispersion inducer composition. The dispersion inducer ofthis invention has a chemical formula as set forth above. Additionalcomponents that may be included in the dispersion inducer compositionsare also set forth above.

The oral composition can be various compositions in the field of dentalhygiene known to those in the art. For instance, the oral compositionmay be a mouthwash, breath spray, dentifrice, tooth powder, whiteningstrips, or prophylaxis paste. As is well known in the art, mouthwashesare commonly used to help remove mucous and food particles in the oralcavity or throat. Mouthwashes typically contain antiseptic and/oranti-plaque components to kill the bacterial plaque that causes caries,gingivitis, and bad breath. They can also contain anti-cavitycomponents, such as fluoride, to protect against tooth decay. Suitablemouthwash components may be found in U.S. Pat. No. 5,968,480, hereinincorporated by reference in its entirety.

Likewise, the same or similar antiseptic, anti-plaque, and anti-cavitycomponents can be used in breath sprays, dentifrices, including geldentifrices, tooth powders, whitening strips, and prophylaxis pastes.Suitable breath spray compositions are disclosed in U.S. Pat. No.7,297,327; suitable tooth powder compositions, such as those used intooth bleaching compositions, are disclosed in U.S. Pat. No. 5,989,526;suitable whitening strips are disclosed in U.S. Pat. No. 6,514,483; andsuitable dentifrices and prophylaxis paste compositions, includingdental abrasives, are disclosed in U.S. Pat. No. 5,939,051, all of whichare herein incorporated by reference in their entirety.

The ingredients of orally-accepted vehicle are similar to thosediscussed above. However, the orally-accepted vehicle will varydepending on the desired consistency and desired end product of the oralcomposition. For instance, a mouthwash is in a liquid form, so liquidcarriers, typically carriers having a high percentage of water, shouldbe used. On the other hand, a gel dentifrice should be in the form of agel and would utilize gelling agents or other carriers that enable thefinal product to be in the form of a gel. The orally-accepted vehicleshould have properties that both allow the dispersion inducercomposition to be delivered while also providing the final product withthe desired consistency.

The oral composition may also be in the form of chewing gum, a breathstrip, a lozenge, or a breath mint. Chewing gum is typically acombination of a water-insoluble phase, or gum base, and a water-solublephase of sweeteners, flavoring and/or food coloring. Other componentsmay also be added to the gum, including breath-freshening additives suchas zinc and phosphate salts, teeth-whitening additives such as silica,and plaque-reducing additives to moderate dental plaque. Suitable gumcompositions may be found in U.S. Pat. Nos. 6,416,744 and 6,592,849,both of which are herein incorporated by reference in their entirety.

Breath strips are similar to chewing gum, except that the strips aredesigned to dissolve in the mouth, often absorbed through the tongue.The strips can deliver bioactive ingredients to freshen the mouth aswell functional bioactive ingredients, such as vitamins, minerals,supplements, pharmaceuticals, and vaccines.

Lozenges and breath mints are typically discoid-shaped solids thatcontain a therapeutic agent in a flavored base. The base may be a hardsugar candy, glycerinated gelatin or combination of sugar withsufficient mucilage to give the composition requisite form. Thedispersion inducer may represent the therapeutic agent, or it may beadded in addition to therapeutic agents known in the art. Suitablelozenge and breath mint compositions are disclosed in U.S. Pat. No.7,025,950, herein incorporated by reference in its entirety.

The oral composition may also be in the form of a cleaning preparationfor a dental apparatus that is placed in the oral cavity. Dentalapparatuses such as dentures, dental dams, and certain types oforthodontic braces are placed in the oral cavity for a period of time,and then periodically removed for cleaning. The cleaning compositionused to clean the dental apparatuses should function in its customarymanner of cleaning the apparatus, but may also contain therapeuticagents that can assist in treating or preventing dental plaque, gingivaldiseases, periodontal diseases, and oral infection when the dentalapparatuses are in contact with the oral cavity. Cleaning compositionssuch as effervescent cleansers made with alkaline mixtures containing achlorine compounds and the like are known in the art. Suitable cleaningcompositions for dental apparatuses are disclosed in U.S. Pat. No.3,936,385, herein incorporated by reference in its entirety. Thedispersion inducer may be added to the cleaning compositions in a mannerthan enables it to coat the dental apparatus upon contact. After thedental apparatus has been introduced into the oral cavity, thedispersion inducer can interact with the teeth and other elements of theoral cavity in a therapeutically effective manner, i.e. to preventdental plaque, gingival diseases, periodontal diseases, and/or oralinfection.

This invention also relates to an article for oral use comprising adental article and a dispersion inducer. The dispersion inducer has thechemical formula set forth above, and is coated on, encapsulated in, orimpregnated in the dental article. Additional components that may beincluded in the dispersion inducer compositions are also set forthabove.

Various dental articles known in the art may used in this embodiment ofthe invention. In one embodiment, the dental article is a dental floss.Any fiber known in the art may be used in the dental floss. Suitablefibers include polyamides (such as nylon), polyesters, polypropylenes,polytetrafluoroethylenes, cellulose, and cotton. Nylon andpolytetrafluoroethylene fibers are the most common fibers used in dentalfloss and represent preferred fibers. Suitable dental flosses aredisclosed in U.S. Pat. Nos. 6,270,890 and 6,289,904, both of which areherein incorporated by reference in their entirety. The dispersioninducer composition may be impregnated into the fiber, coated on thefiber, or otherwise incorporated into the dental floss.

The dental floss may be coated or impregnated with a wax or otherhydrophobic substance for ease of use during the flossing process.Suitable waxes include microcrystalline waxes, beeswax, paraffin waxes,carnauba waxes, and polyethylene waxes. The dispersion inducercomposition may be coated onto the dental floss as part of the waxlayer, as a second or additional layer in conjunction with the waxlayer, or applied to the fiber as discussed above.

The dental article may be a toothpick that is impregnated with or coatedwith the dispersion inducer composition. Toothpicks may be made fromnatural products, such as wood, or artificial components, includingvarious plastics. Suitable toothpicks are disclosed in U.S. Pat. No.7,264,005, herein incorporated by reference in its entirety.

The dental article may also be a dental appliance such as a dentalaspirator, bite block, dental dam, tongue stabilizer, tongue deflector,or any other piece of dental equipment that a dentist or dentalassistant may use in the mouth of a patient. A discussion of dentalappliances may be found in U.S. Pat. Nos. 4,865,545 and 5,152,686, bothof which are herein incorporated by reference. The portion of the dentalappliance that comes into contact with the oral cavity of a patient maybe coated with the dispersion inducer composition.

The dental article may also be a dental construct, such as a veneers,crowns, inlays, onlays, or bridges that are placed on the teeth. Dentalconstructs are typically made of metal alloys, porcelain, ceramic,amalgam, acrylate polymers, or a combination of these materials.Suitable dental constructs are disclosed in U.S. Pat. No. 7,229,286,herein incorporated by reference in its entirety. The dispersion inducercomposition may be embedded in the composition used to make the dentalconstruct. Alternatively, the dispersion inducer composition may becoated on the dental construct after it has been prepared.

This invention also relates to an aqueous composition applied to theoral cavity with the use of a dental article, comprising water and adispersion inducer composition. Various dental articles are attached toor designed to be used with a water line so that water can bedistributed through the dental article, and then routed from the dentalarticle to the oral cavity of a subject. Suitable dental articlesinclude dental water lines, dental water picks, and the like.

While tap water or purified water may be used in these type of dentaldevices, the water source may also be supplemented with additives sothat the water delivers the additives to the oral cavity of the subjectwhen used with the dental article. In this case, the additivesupplemented to the water is a dispersion inducer composition.

Dental water lines and dental water picks are known in the art andcommonly used by dentists and dental assistants. A discussion ofdifferent types of dental water lines and their different applicationsmay be found in U.S. Pat. No. 5,785,523, herein incorporated byreference in its entirety. Suitable water picks are disclosed in U.S.Pat. No. 4,257,433, herein incorporated by reference in its entirety.

The present invention also relates to a method of cleaning and/ordisinfecting contact lenses. The method involves treating contact lenseswith a cleaning and/or disinfecting solution containing the dispersioninducer according to the present invention. The contact lens may betreated in this manner while being stored in solution or while beingused in vivo. Alternatively, the dispersion inducer can be used in eyedrops.

The present invention further relates to a method of treating and/orpreventing acne or other biofilm-associated skin infections on the skinof a subject. The method involves treating the skin of the subject withthe dispersion inducer according to the present invention underconditions effective to treat and/or prevent the acne orbiofilm-associated skin infections. The dispersion inducer may bepresent in an ointment, cream, liniment, salves, shaving lotion, oraftershave. It may also be present in a powder, cosmetic, ointment,cream, liquid, soap, gel, cosmetic applicator, and/or solid, woven ornon-woven material intended to contact or be proximate with the skin.

The present invention also relates to a method of treating and/orpreventing a chronic biofilm-associated disease in a subject. The methodinvolves administering to the subject the dispersion inducer accordingto the present invention under conditions effective to treat and/orprevent the chronic biofilm-associated disease. The chronicbiofilm-associated diseases to be treated and/or prevented include, butare not limited to, middle ear infections, osteomyelitis, prostatitis,colitis, vaginitis, urethritis, arterial plaques, sinovial infections,infections along tissue fascia, respiratory tract infections (e.g.,infections associated with lung infections of cystic fibrosis patients,pneumonia, pleurisy, pericardial infections), genito-urinary infections,and gastric or duodenal ulcer infections. For gastric or duodenal ulcerscaused by Helicobacter pylori, the dispersion inducer will need tofunction at a pH of below 5.5. The dispersion inducer may beadministered in combination with an antimicrobial agent, such asbiocides, surfactants, antibiotics, antiseptics, detergents, chelatingagents, or virulence factor inhibitors. In the case of gastrictherapies, acid reducing therapies, such as antacids, proton pumpinhibitors, antihistamines, and the like may also be employed.

Another aspect of the present application relates to a solutioncomprising:

a dispersion inducer comprises:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 4 to 7, andR is a carboxylic acid, where said inducer is present at a concentrationless than 0.5 percent by weight, and where said solution has a pHgreater than 5.

A further aspect of the present invention is directed to a compositioncomprising: a component selected from one or more of the groupconsisting of biocides, surfactants, antibiotics, antiseptics,detergents, chelating agents, virulence factor inhibitors, gels,polymers, pastes, edible products, and chewable products. In addition,the composition includes a dispersion inducer comprises:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 4 to 7, andR is a carboxylic acid. The inducer is formulated in a non-salt form.

The present invention is also directed to a solution which includes acis isomer of 2-decenoic acid, where the solution is selected from thegroup consisting of a skin cream, a toothpaste, and a mouthwash andwhere the solution is substantially free of the trans isomer of2-decenoic acid. As interpreted herein, this solution is substantiallyfree of a trans isomer if a reduction in trans isomer (without change incis-isomer) concentration does not increase bioactivity. It is morepreferred that there be a molar ratio of cis to trans of at least 2.

Another form of the present application is directed to a solutioncomprising: a cis isomer of 2-decenoic acid, where the solution isselected from the group consisting of a skin cream, a toothpaste, and amouthwash and where said solution is trans isomer-free.

Another form of the present application is for a method which comprisesproviding contact lenses and a solution comprising a dispersion inducerat a concentration less than 0.5% by weight, said inducer comprises:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 4 to 7, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid. The contactlenses are then treated with said solution.

A further form of the present invention is for a method which involvesproviding a subject with a skin condition and a solution having a pHgreater than 5, where the solution comprising a dispersion inducer at aconcentration less than 0.5% by weight said inducer comprises:H₃C—(CH₂)_(n)—CH_(m)

CH_(m)R,where

is a single or double carbon-carbon bond, m is 1 or 2, n is 4 to 7, andR is a carboxylic acid, a salt, an ester, or an amide, where the esteror amide is an isostere or biostere of the carboxylic acid. The skincondition is then treated with the solution.

The present invention also relates to a composition comprising one ormore dispersion inducers and one or more additive components. Theseadditive components are selected from the group consisting of biocides,surfactants, antibiotics, antiseptics, detergents, chelating agents,virulence factor inhibitors, gels, polymers, pastes, edible products,and chewable products. The composition is formulated so that when it iscontacted with a biofilm produced by a microorganism, where the biofilmcomprises a matrix and microorganism on a surface, the dispersioninducer selectively acts on the microorganism and has a suitablebiological response without a required direct effect to disrupt thematrix.

Another aspect of the present invention relates to a method of treatingor preventing a condition mediated by a biofilm in a subject. Thismethod involves providing a subject having, or susceptible to, acondition mediated by a biofilm produced by a microorganism, whereby thebiofilm comprises a matrix and the micro-organism on a surface. Adispersion inducer is administered to the subject under conditionseffective for the dispersion inducer to selectively act on themicroorganism and have a suitable biological response without a requireddirect effect on the matrix, whereby the condition mediated by a biofilmin the subject is treated or prevented.

A further embodiment of the present application is directed to a methodof treating or inhibiting formation of a biofilm on a surface. Thisinvolves providing a surface having or being susceptible to formation ofa biofilm produced by a microorganism, whereby the biofilm comprises amatrix and the micro-organism on the surface. A dispersion inducer isadministered to the surface under conditions effective for thedispersion inducer to selectively act on the microorganism and have asuitable biological response without a required direct effect on thematrix, whereby formation of the biofilm on the surface is treated orinhibited.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1 Bacterial Strains and Media

The microorganisms used in this study included Pseudomonas aeruginosaPAO1 from B. H. Holloway, Escherichia coli (ATCC 10798), Proteusmirabilis (ATCC 25933), Klebsiella pneumoniae (ATCC 10273),Staphylococcus aureus (ATCC 12602), Streptococcus pyogenes (ATCC 19615),Bacillus subtilis (ATCC 6633), and Candida albicans (ATCC 20260) and amixed undefined culture collected on R2A plates via airbornecontamination Except where indicated, all experiments were performed inmodified EPRI medium, containing 0.005% ammonium nitrate, 0.00019%KH₂PO₄, 0.00063% K₂HPO₄ (pH 7.0), and 0.001% Hutner salts (Cohen-Bazireet al., J. Cell. Comp. Physiol. 49:35 (1957), which is herebyincorporated by reference in its entirety), supplemented with 0.2%glucose. C. albicans was grown in modified EPRI medium supplemented with0.2% glucose and 0.1% peptone. K. pneumoniae, P. mirabilis, S. aureus,and B. subtilis were grown in modified EPRI medium supplemented with0.1% peptone. S. pyogenes was grown in 10% Brain Heart Infusion broth.

Example 2 Preparation of P. aeruginosa Spent Medium

To prepare cell-free spent culture medium, 6 mL of an overnight cultureof P. aeruginosa PAO1 grown in modified EPRI medium at 30° C. wereinoculated into four liters of modified EPRI medium and incubated for 10days at room temperature with continuous stirring. Bacterial cells weresedimented by centrifugation (Sorvall RC 5B Plus Centrifuge, GSA Rotor;Thermo Electron Co., Ashville, N.C.) at 13,000×g for 15 minutes at 4° C.The supernatant was removed and filtered under vacuum through a 0.45 μmMillipore Type HA filter (Millipore. Co., Billerica, Mass.) andsubsequently, through a 0.2 μm, Acrodisc 32 mm syringe filter (PALL Co.,East Hills, N.Y.). Spent medium was stored at 4° C.

Example 3 Preparation of CSM

The organic components of spent medium were extracted by adding 80 mL ofchloroform to 250 mL of filtered spent medium in a separatory funnel.The chloroform fraction was removed after a separation time of 1 hr.Chloroform was evaporated at 40° C. using a Rotavapor R-3000 rotaryevaporator (Büchi Laboratories, Flawil, Switzerland) and the remainingorganic material was re-suspended in 6 mL of filtered nanopure water andevaporated to dryness using a Speed-Vac evaporator system (SavantInstruments, Inc., Hicksville, N.Y.) or lyophilized. These samples werethen resuspended in culture medium or purified water. The final productis referred to as Chloroform extracted Spent Medium (CSM). Except whereindicated, CSM was used in experiments at a final chloroform extractedorganic carbon concentration 125 fold greater than found in spentmedium.

Example 4 HPLC Fractionation of CSM

CSM was fractionated by High Performance Liquid Chromatography (HPLC)(Varian Prostar model 320, Varian Inc., Palo Alto, Calif.) using a C18Microsorb-mv reverse phase column (Varian Inc.) dimensions 250×4.6 mm.The column was loaded with 100 μL of CSM and eluted in anacetonitrile/water gradient (2-75%) with a flow rate of 1 mL×min⁻¹ for29 minutes. Samples were collected every minute, starting at 2 minutes.HPLC fractions were pooled and concentrated in a Speed Vac concentrator(Savant Instruments, Inc., Hicksville, N.Y.) and resuspended in 0.5 mLof modified EPRI medium or purified water. The active HPLC fraction wasfound to elute from the column in 70% acetonitrile/30% water. The activefraction of each HPLC separation was determined by microtiter platedispersion bioassay.

Example 5 Microtiter Plate Dispersion Bioassay

Microtiter plate dispersion bioassays were used to test variouspreparations for their ability to exogenously induce biofilm dispersion.Biofilms were grown on the inside surface of microtiter plate wellsusing a semi-batch culture method in which the medium within each wellwas replaced periodically to reduce the accumulation of nativedispersion inducing factors. Biofilms grown in this manner were treatedwith dispersion inducer or sterile medium to release cells into the bulkliquid and evaluate dispersed cell number by measuring optical density.Briefly, sterile polystyrene 96 well plates were etched with acetone for10 seconds to create a rough surface for the attachment of microbialcells. After drying for 24 hours, plates were inoculated with 150μL/well of overnight culture containing the test organism, previouslydiluted 1:20 in growth medium and incubated at 30° C. with shaking at200 rpm. Medium in the wells was replaced every 24 hours for 5 days andevery 12 hours on day 6 and day 7. Medium was then replaced after 7hours. Dispersion induction was tested by adding 150 μL growth mediumcontaining dispersion inducer for 1 hr at 30° C. or sterile medium as acontrol. Medium containing dispersed cells was then transferred by pipetto a non-etched microtiter plate and the optical density (OD₅₇₀)Winooski, Vt.). Treatments consisted of spent medium, CSM,cis-2-decenoic acid, was determined (ELx808 Absorbance MicroplateReader; BioTek Instruments, Inc., trans-decenoic acid, decanoic acid andDSF at various concentrations. Ethanol (10%) was used as a carrier forfatty acid inducer samples and was determined to have no influence ondispersion. Results from use of this method are meaningful in makingcomparisons of different treatments and to determine whether dispersionactivity is statistically significant. Note: Microtiter plate dispersionbioassays were not suitable for determining absolute magnitude of aninduced dispersion response because in a semi-batch system, control andtest samples are susceptible to natural dispersion against which theactivity of exogenous induction is measured. All efficiency studies wereperformed using biofilm tube reactor or flow-cell continuous culturesystems and were based on both total cell counts and viable cell counts.

Example 6 Dispersion Bioassays in Biofilm Tube Reactors

P. aeruginosa PAO1 biofilm cultures were grown in tube reactors asdescribed previously by Sauer et al. (K. Sauer, et al., J. Bacteriol.184:1140 (2002), which is hereby incorporated by reference in itsentirety). A continuous once-through tube reactor system was configuredusing 8 silicone reactor tubes (81.5 cm length×14 mm ID), connected toan 8-roller head peristaltic pump and medium reservoir, via additionalsilicone tubing. Medium was pumped through the tubing to a closedeffluent medium reservoir. The assembled system was sterilized byautoclaving prior to inoculation. The silicone tubes were inoculated bysyringe injection through a septum 1 cm upstream from each reactor tube,with 2 mL of overnight cultures of P. aeruginosa (containingapproximately 1×10⁸ CFU mL⁻¹). Bacterial cells were allowed to attach(static incubation) to the tubing for 1 hour, after which the flow wasstarted at an elution rate of 10.8 mL hr⁻¹. Treatments were carried outfollowing 96 hours of P. aeruginosa PAO1 biofilm cultures. Thetreatments were performed under continuous and static conditions.

Under continuous treatment (FIG. 17A) the influent medium was changedfrom fresh medium in the test lines to spent medium amended with 2%glucose, adjusted to neutrality and aerated overnight prior to additionControl lines were switched to new lines containing fresh modified EPRImedium. Samples were collected for one minute intervals starting attime=0 min, and assayed for optical density. Spent medium was added attime=30 min. Samples were collected in test tubes on ice and weresubsequently homogenized for 30 sec at 5000 rpm with a Tissue TearorModel 985370 (Biospec Products, Inc.) to ensure separation of cells.Cell density was determined by optical density at 600 nm with anUltrospec 3000 spectrophotometer (Amersham Pharmacia Biotech, Inc.).

Under conditions of static treatment, dispersion inducer was added bysyringe injection through the inoculation port, 2 cm upstream from thebeginning of the tube reactor, displacing the reactor volume with mediumcontaining inducer. Spent medium was added directly. CSM or syntheticdispersion inducer (eg: cis-2-decenoic acid) was prepared in modifiedEPRI and added. After one hour of exposure under non-flowing conditions,an 81.5 cm length of each silicone tube reactor was cut out, the liquidfraction (containing released biofilm cells) was collected in test tubeson ice and the biofilm fraction was collected by rolling the tube on thelab bench with a metal rod to extrude the cells remaining in the lumenof the tube (K. Sauer, et al., J. Bacteriol. 184:1140 (2002), which ishereby incorporated by reference in its entirety). Samples werecollected on ice, and homogenized as above. Cells numbers weredetermined by spread plate method on Standard Plate Count agar medium(Difco, Detroit, Mich.) or by optical density at OD₆₀₀ adjusted to cellnumber by calibration to a standard curve for cell number as determinedmicroscopically by total cell count. Dispersion efficacy was calculatedusing either optical density or viability measurements:

${{Dispersion}\mspace{14mu}{Efficacy}} = \frac{{Cells}\mspace{14mu}{from}\mspace{14mu}{Bulk}\mspace{14mu}{Liquid} \times 100}{\begin{matrix}{{{Cells}\mspace{14mu}{from}\mspace{14mu}{Bulk}\mspace{14mu}{Liquid}} +} \\{{Cells}\mspace{14mu}{from}\mspace{14mu}{Biofilm}}\end{matrix}}$

Example 7 Microscopic Analysis

A continuous-culture once-through flow cell was configured to observethe growth and development of biofilms attached to a glass substratum.The flow cell was constructed of aluminum containing a chamber 1.0 mm by1.4 cm by 4.0 cm capped with a glass cover slip. Sterile modified EPRImedium was pumped from a 10-liter vessel through silicone tubing to theflow cell using a Masterflex 8-roller-head peristaltic pump at a flowrate of 0.13 mL min⁻¹. Flow through the chamber was laminar, with aReynolds number of 0.17, having a fluid residence time of 4.3 min.Medium leaving the flow cell was discharged to an effluent reservoir viasilicone tubing. The entire system was closed to the outside environmentbut maintained in equilibrium with atmospheric pressure by a0.2-μm-pore-size gas-permeable filter fitted to each vessel. Log-phaseP. aeruginosa (approximately 10⁸ CFU mL⁻¹) were inoculated as a 3.0 mLslug dose through a septum 4 cm upstream from the flow cell underflowing conditions. Cells attached to the inner surface of the glasscover slip were viewed by transmitted light or epi-UV illumination usingan Olympus BX60 microscope and a 100× magnification A100PL objectivelens or a 50× magnification ULWD MSPlan long working distance Olympusobjective lens. All images were captured using a Magnafire cooledthree-chip charge-coupled device (CCD) camera (Optronics Inc., Galena,Calif.) and stored as separate digital files for subsequent retrievaland analysis P. aeruginosa were grown in the flow cell for up to 12days. Previous work by applicant has shown P. aeruginosa to developsteady-state biofilms following a continuous culture period of 7 to 9days. Steady state is defined by no change in effluent cell counts (CFU)resulting from detached biofilm cells; in steady state, growth of thebiofilm is balanced by the loss of cells through dispersion ordetachment. Individual cell clusters were examined during the course ofeach experiment and assigned grid coordinates, which were reexaminedperiodically during the course of the experiments. Size measurementswere taken of random cell clusters by locating the cluster nearest to arandomly selected microscope stage coordinate. Each cell cluster wasmeasured to determine its height by focusing from the substratum throughto the apex of the cluster, and its width by measurement at the base ofthe cell cluster using a stage micrometer. Cell clusters were defined ascells embedded within an exopolysaccharide matrix attached to thesubstratum and lacking motility; void areas within cell clusters weredetermined by the observation of free-swimming bacteria within a spaceinside a cell cluster.

Example 8 Inhibition of Biofilm Development

A flow cell was used to culture bacteria on the surface of a glasssubstratum (described above). Biofilms of P. aeruginosa were grown atroom temperature over a period of 99 hours in the presence and absenceof CSM (diluted 1:125 to match the concentration of the chloroformextracted organic material found in spent medium) in modified EPRImedium. During the course of the experiment, the total cell coverage ofthe bacteria on the surface and average biofilm thickness weredetermined by counting 20 microscope fields using a 50×ULWD MSPlanobjective lens for each time point. Using the image analysis software,ImagePro Plus, the total area of cells per cm² was determined at 72hours and 99 hours. Thickness was determined by measuring the averagemaximum height of 20 random cell clusters at 72 hours and 99 hours ofgrowth. Control samples were grown and tested in the presence ofmodified EPRI medium with no added CSM. Results from these experimentsshowed that surface area coverage of the growing biofilm wassignificantly reduced when biofilms were grown in the presence of CSMcompared to biofilms grown in EPRI medium alone. The addition of CSMalso caused a significant reduction in the average biofilm cell clusterthickness after 99 hours growth, compared to samples not treated withCSM (FIG. 18).

Example 9 Spectral Analysis of P. aeruginosa CSM and Cis-2-Decenoic Acid

All CSM samples prepared in purified water were lyophilized andresuspended in appropriate carriers for each spectroscopic analysis. CSMcontrols in all experiments consisted of CSM HPLC products that did notinduce dispersion as determined by microtiter plate dispersion bioassay,and carrier solution not containing CSM.

Example 10 Mass Spectroscopy

Samples were resuspended in carrier solution (50% water, 50% methanoland 0.01% formic acid). Mass spectroscopy was performed using ahigh-performance, hybrid quadrupole time-of-flight massspectrometer—QSTAR® XL Hybrid LC/MS/MS System (Applied Biosystems,Foster City, Calif., USA)—in positive ion mode, at room temperature,with an IonSpray source for API 150EX™, API 3000™ and QSTAR® Systems(Applied Biosystems). Data were analyzed using Analyst QS version 1.1.

Example 11 Nuclear Magnetic Resonance (NMR)

Samples of CSM and cis-2-decenoic acid were resuspended in 1 mL ofdeuterated acetonitrile and inserted into a thin walled NMR sample tube(VWR). Analyzes was performed in a 300 MHz Proton NMR-Bruker AC 300(Bruker Daltonics Inc., Vilarica, Mass., USA). Spectra were accumulatedfor 24 hours.

Example 12 Gas Chromatography-Mass Spectroscopy (GC-MS)

Samples of CSM and concentrations of cis-2-decenoic acid from 0.01mg×mL⁻¹-10 mg×mL⁻¹ were resuspended in 2 mL of acetonitrile. A 3step-sequential hexane extraction was performed to remove solubleorganic sample material. Hexane was evaporated to dryness in a waterbath (55-70° C.). Puridine (250 μL) was subsequently added to solubilizesamples for injection into GC. Spectra were obtained with a ShimadzuQP5050A GC-MS system, using helium as a carrier as and a Restek(Columbia, Md.) XTI-5 GC column (30 m, 0.25 mm i.d., 0.25 μm filmthickness) with a 1 mL×min⁻¹ flow rate. All analyses incorporatedsplitless injection and electron impact ionization. The interfacetemperature between the GC and the MS was maintained at 310° C. Datawere analyzed using the program Lab Solutions, GCMS solution version1.2.

Example 13 Infrared Spectroscopy (IR)

Samples of CSM and cis-2-decenoic acid were weighed before and afterlyophilization to determine the amount of KBr to add to each sample. KBrwas added at 10 times the sample mass and mixed using a mortar andpestle. The resulting powder was formed into a pellet using a Carver4350 Manual Pellet Press (Carver Inc., Wabash, 1N, USA). Pressure wasapplied at 10 Tons for 10 min. IR spectra were obtained using a BrukerEquinox 55 FT-IR spectrometer at room temperature in the range of 3500cm⁻¹ to 400 cm⁻¹ at a resolution of 1 cm⁻¹. The final spectra representthe mean of 128 scans. Each sample was measured in triplicate.

Example 14 Biofilm Bacteria are Resistant to Antibiotics

FIG. 1A illustrates schematically how biofilm bacteria are resistant tothe addition of antibiotics, with similar resistance shown for biocidesand other antimicrobial treatments. FIG. 1B illustrates that if adispersion inducer is added in addition to antibiotic, the dispersedbacteria lose their resistance and become susceptible to the antibiotic.

Example 15 Effect of Dispersion Inducing Compounds

FIG. 2 shows an actual biofilm sample treated with the dispersioninducing compound according to the present invention, derived fromcultures of Pseudomonas aeruginosa. In this experiment, a once-throughflow-cell was used to culture P. aeruginosa over a period of six daysprior to testing with added CSM.

The flow cell was constructed of anodized aluminum, containing a chamber1.0 mm by 1.4 cm by 4.0 cm capped with a glass cover slip. Sterile EPRImedium was pumped from a 2-liter vessel through silicone tubing to theflow cell using a Masterflex 8-roller-head peristaltic pump at a flowrate of 0.13 ml min⁻¹. Flow through the chamber was laminar, with aReynolds number of 0.17, having a fluid residence time of 4.3 min.Medium leaving the flow cell was discharged to an effluent reservoir viasilicone tubing. The entire system was closed to the outside environmentbut maintained in equilibrium with atmospheric pressure by a0.2-μm-pore-size gas-permeable filter fitted to each vessel. Log-phaseP. aeruginosa (approximately 10⁸ CFU/ml) were inoculated as a 3.0-mlslug dose through a septum 4 cm upstream from the flow cell underflowing conditions. Cells attached to the inner surface of the glasscover slip were viewed by transmitted light using an Olympus BX60microscope and a 50× magnification ULWD MSPlan long working distanceOlympus objective lens. All images were captured using a Magnafirecooled three-chip charge-coupled device (CCD) camera (Optronics Inc.,Galena, Calif.) and stored as separate digital files for subsequentretrieval and analysis. Following development of a mature biofilm withinthe flow-cell, medium-flow was stopped and 3 mL of filtered CSM insterile EPRI medium was added to the flow-cell. Transmitted light imagesof a single location within the flow-cell were taken before and duringtreatment with CSM. FIG. 2 shows images taken from such an experiment 1min prior to addition of CSM, 5 min after addition of CSM, and 30 minafter addition of CSM. Control samples were also run in the same manneras the test samples with the exception that CSM was not included withthe 6 mL of added EPRI medium. Results from the control samples showedno change in biofilm cell numbers or biofilm architecture, with nodispersion evident.

Example 16 Biofilm Bacteria Undergo a Phenotypic Switch

During the course of normal biofilm development, biofilm bacteriaundergo a phenotypic switch at the end of maturation II stage (FIG. 3)in which their physiology changes from a predominantly biofilm form to apredominantly planktonic form. Microscopic observations of biofilmsduring the dispersion phase demonstrated that bacteria within cellclusters become motile (maturation stage P. aeruginosa are non-motile),while the bacteria around the edges of the clusters remain fixed. Theregion of the cell cluster within which bacteria can swim/twitch growsin volume from a (usually) central location and eventually a breach ismade in the cluster wall. The bacteria are able to swim through thisbreach and enter the bulk liquid phase leaving behind a void within thecell cluster.

Continued study of the dispersion response has revealed that cellclusters transition through episodes of growth and dispersion; the samecell cluster often enduring many such cycles. Multiple dispersion andregrowth events generally lead to the development of cell clusters withpatterns analogous to growth rings which can indicate the number oftimes that dispersion has occurred. Often cell clusters will detach fromthe substratum completely during a dispersion event (Stoodley et al.,“Growth and Detachment of Cell Clusters from Mature Mixed-speciesBiofilms,” Appl. Environ. Microbiol. 67:5608-5613 (2001), which ishereby incorporated by reference in its entirety). This effect isthought to be due to weakening of attachment structures at the base ofthe cell cluster allowing fluid sheer to detach the cluster.

Example 17 Cell Detachment after Medium Stagnation

FIG. 4 depicts a time series of phase contrast photomicrographs showingthe detachment of cells after medium stagnation of 72 hours. Flow-cellwere inoculated with P. aeruginosa PAO1 and cultured for a period ofthree days, according to the method described in Example 3, above. Thechoice of three days for culture of these biofilms was based upon theobservation that under continuous flow, cell clusters within a biofilmof P. aeruginosa were observed to undergo spontaneous dispersion eventsfollowing 9 days of incubation. After 72 days of growth under continuousflow, medium flow was stopped and images of the cell clusters wererecorded every two hours for a period of 96 hours. After 72 hours ofmedium stagnation, the cell clusters within the flow-cell were observedto dis-aggregate, with cells entering the bulk liquid medium asplanktonic bacteria (FIG. 4). These experiments demonstrated thatcessation of flow induced dispersion of biofilms response. In theseexperiments, dispersion occurred not simply within the cell clusters,but throughout all clusters in the biofilm. Only those cells which weredirectly attached to the substratum were not observed to swim into thebulk liquid, as illustrated in FIG. 4.

Example 18 Development of Chloroform Extraction Method

Various growth and extraction procedures were tested to develop areliable method of extracting the active fraction of spent culturemedium having dispersion inducing activity. Chloroform was chosen as theextraction solvent of choice due to its compatibility with HPLCfractionation procedures, because it resulted in a narrow range ofextractable organic compounds (as determined by mass spectrometry) andbecause it could recover bioactive amounts of the dispersion inducingagent. The method currently used for chloroform extraction of spentmedium follows: Bacterial cultures of P. aeruginosa PAO1 were grown in 4liters of EPRI medium (containing: sodium lactate 0.05 g/l, sodiumsuccinate 0.05 g/l, ammonium nitrate 50.381 g/l, KH₂PO₄ 0.19 g/l, K₂HPO₄0.63 g/l, Hutner Salts metals solution 1 ml, and glucose, 2.0 g/l) in abatch culture vessel for six days at room temperature with continuousstirring. Following growth, bacteria were removed from the culturemedium by centrifugation at 10,000×g for 20 min, followed by filtrationof spent medium through a 0.22 μm pore size filter. In batches, 250 mlof filtered spent medium were mixed with 80 ml of chloroform in aseparatory funnel. The chloroform fraction was removed after aseparation time of 10 min. The chloroform samples were then evaporatedto dryness at 70° C. using a rotavapor R-3000 (Biichi Laboratories,Flawil, Switzerland) and re-suspended in 6 mL of filtered nanopure wateror EPRI medium. The final product, resulting from the chloroformextraction procedure is referred to here as concentrated spent medium orCSM. FIG. 15 shows the results of comparing the effect of CSM and SpentMedium on continuous culture biofilms grown in biofilm tube reactors.CSM and Spent medium were prepared as described previously from culturesof P. aeruginosa PAO1 grown at 22° C. for 9 days in EPRI mediumsupplemented with 2.0 gram per Liter of glucose. Biofilms of P.aeruginosa PAO1 were cultured for six days at 22° C. in biofilm tubereactors consisting of 32 cm silicone dioxide Masterflex size 14 tubing.At the end of six days, 6 mL of CSM, Spent Medium or Sterile EPRImedium, each supplemented with 2.0 gram per Liter glucose, was added tothe tubes. The effluent from the tubes was collected and pooled for eachtreatment over a period of 20 minutes. Pooled sampled were assayed foroptical density at 570 nm to determine relative cell numbers dispersedfrom each treatment. Each experiment was performed with five replicates.Results from these experiments demonstrated that chloroform extractedspent culture medium, CSM showed a greater activity in dispersingbiofilms of P. aeruginosa compared to spent medium.

Applicant observed that P. aeruginosa PAO1 will disperse from acontinuous culture biofilm grown on a glass substratum in a flow-cellreactor after medium flow had been stopped for several hours. Thisobservation has led to the hypothesis that biofilm dispersion may resultfrom the accumulation of an extracellular messenger which acts as aninducer of biofilm disaggregation. This hypothesis is supported byobservations that biofilms of P. aeruginosa will not form in batchculture flasks, but will form on the walls of a chemostat, indicatingthat accumulation of a signal for dispersion may prevent biofilmdevelopment. Furthermore, when grown in continuous culture,microcolonies of P. aeruginosa will form hollow voids at their centerwhen they attain a minimum diameter of 40 microns and thickness of 10microns (FIG. 6). The microcolony size within which these voids form,however, is dependent on the fluid flow rate. When flow in a biofilmreactor was increased, the diameter and thickness at which microcolonyvoid formation occurred also increased, indicating a relationshipbetween dispersion induction and transport. These observations hintedthat an extracellular substance produced by P. aeruginosa wasresponsible for inducing biofilm dispersion.

If P. aeruginosa produces an extracellular dispersion-inducing compound,applicant postulated that the addition of cell-free spent culture mediumto mature P. aeruginosa biofilms should cause the release of cells intothe bulk liquid medium. These bacteria should be detectable as anincrease in the number of cells recovered in the reactor effluent. FIG.7A illustrates results from a representative experiment in whichbiofilms were treated for 70 minutes under continuous flow withcell-free spent medium in which P. aeruginosa had been grown insuspension for 24 hours; fresh medium was added to control biofilms.Prior to addition, spent medium was aerated, supplemented with glucoseand its pH adjusted to neutrality, to ensure that starvation, oxygendepletion or a change in pH was not responsible for the release ofbacteria A large spike in the effluent cell number was detectablecompared to control lines within 20 minutes of the addition of the spentmedium, indicating the release of biofilm bacteria into the effluent ofcultures treated with spent medium A small spike of released cells wasalso detectable in control samples, likely representing a response tothe physical or mechanical effects associated with switching lines to afresh medium reservoir.

To purify the active dispersion inducing fraction of spent medium,cell-fee stationary-phase batch cultures of P. aeruginosa were extractedusing chloroform, followed by rotary evaporation of the chloroform andre-suspension of the organic fraction in fresh medium or buffer solution(resulting in a 125-fold increase in the chloroform-soluble organicfraction). This preparation is referred to as CSM. To test thedispersion-inducing activity of CSM, P. aeruginosa biofilms were grownin continuous culture in silicone tubing and exposed the biofilms forone hour to medium amended with CSM. The extruded contents of the tubereactors showed a largely intact biofilm in the control line treatedwith fresh medium (FIG. 7B), while the contents of the tubes treatedwith CSM showed the biofilm to have completely disaggregated (FIG. 7C).Studies of 4-day old biofilms grown in continuous culture in siliconetubing revealed that treatment with CSM-containing medium for one hr waseffective in releasing an average 87.4% (±1.4%) of biofilm cells asdetermined by colony forming units released into the effluent. Spentmedium was shown to have an average dispersion efficacy of 32.4%(±5.5%).

Microscopy was used to evaluate the effect of CSM on biofilmmicrocolonies grown for six days in continuous culture on the glasssubstratum of a flow-cell mounted to a microscope (K. Sauer et al., J.Bacteriol. 184:1140 (2002), which is hereby incorporated by reference inits entirety). Prior to the addition of CSM, a well-developedmicrocolony was observed to contain cells that were stationary andshowed no sign of motility (FIG. 7D). Following 7 minutes of contactwith CSM-containing medium, cells within the microcolony began to twitchand display active motility (FIG. 7E). After 30 minutes, the microcolonyhad become completely disaggregated and cells were observed to swimfreely through the medium (FIG. 7F). When compared to naturaldispersion, exogenously induced dispersion was observed to progress fromthe outside of the microcolony towards the interior and, instead ofcreating a central void, resulted in complete disaggregation of themicrocolony.

When added continuously to flow cells, CSM adjusted to the concentrationof spent medium, showed a significant inhibition of biofilm developmentover a period of 99 hr, demonstrating a reduction in both biofilmaverage thickness and surface area coverage (FIG. 8). Exogenousdispersion induction of pre-formed biofilms by CSM was measurable at alltime points from day 1 (beginning of biofilm microcolony formation)through day 6, after which natural dispersion began to occur. Activityof CSM was shown to persist up to 6 months with no significant reductionwhen stored under refrigeration. Extraction of spent medium by ethylacetate (to recover acyl-homoserine lactones) did not result in apreparation with dispersion activity.

Having demonstrated dispersion induction against mature and developingbiofilms formed by P. aeruginosa, the ability of CSM to inducedispersion in biofilm cultures of E. coli, E. coli mixed with P.aeruginosa, an undefined mixed bacterial biofilm derived from airbornecontaminants, and against biofilms formed by Klebsiella pneumoniae,Proteus mirabilis, Streptococcus pyogenes, Bacillus subtilis,Staphylococcus aureus, and Candida albicans was next tested. CSM wasshown to stimulate significant dispersion compared to controls in allsamples tested. Results from these experiments are summarized in FIG. 9.The ability of P. aeruginosa dispersion inducer to activate dispersionin different species of bacteria and in yeast indicates that itpossesses cross-phylum and cross-kingdom activity.

Having established the role of CSM as an inducer of biofilm dispersion,the active molecule or molecules present in CSM was identified. Thisbegan by assaying the dispersion activity of multiple fractions of CSMseparated by Isocratic gradient in acetonitrile and water using C-18reverse phase high performance liquid chromatography (HPLC). Eluted HPLCfractions (collected at one minute intervals) were desiccated in aSpeedvac to remove residual acetonitrile and re-suspended in purifiedwater and tested by microtiter plate dispersion bioassay to determinedispersion activity. FIG. 10A shows the results of CSM fractionationbiofilm dispersion assays. The results indicated that the HPLC fractionof CSM showing the highest activity eluted at 22 min, anacetonitrile/water ratio of 70%/30%.

Mass spectrometry of the active HPLC CSM fraction showed a consistentmolecular peak with low ionization activity at 171 M/Z (mw=170). Thispeak was present in all samples showing dispersion activity and missingfrom all samples lacking dispersion activity. This peak was also shownto be missing from all carrier liquids and solvents used in preparingCSM (including fresh culture medium). Mass spectroscopy-product ionanalysis of the 170 mw peak, solubility analysis, H¹- and C¹³ nuclearmagnetic resonance (NMR) spectroscopy and infra-red (IR) spectroscopyhave demonstrated that the 170 mw molecule was a mono-unsaturatedC₁₀-fatty acid, with a double bond located at the number 2 carbon;2-decenoic acid.

In order to confirm that the 170-mw molecule (M/Z=171) from the22-minute CSM HPLC fraction was identical to 2-decenoic acid, theoriginal molecule was fragmented in the mass spec to generate production peaks. The product ions from the active CSM fraction and 2-decenoicacid were analyzed by quadrapole ms/ms to evaluate cleavage differencesbetween these two molecules. FIG. 12A, shows that the 171 M/Z CSM samplehad identity with 2-decenoic acid. When analyzed by GC-MS,unfractionated CSM displayed a single major peak with a retention timeof 7.6 min identical to that of 2-decenoic acid FIG. 12B. Infraredspectroscopy confirmed that the cis isomer of 2-decenoic acid was theorganic compound isolated from CSM, FIG. 12C.

Following this identification, mono-unsaturated fatty acid molecules ofvarious molecular weights were synthesized and tested these fordispersion activity. DSF, which was shown to disrupt cell flocs of X.campestris was shown not to promote dispersion of P. aeruginosa. Thecompounds having the highest activity were two isomers of 2-decenoicacid. The trans isomer (trans-2-decenoic acid) was shown by microtiterplate dispersion bioassay to have activity only at millimolarconcentrations, typically not low enough to qualify as a cell-cellsignaling molecule. FIG. 10B shows the dispersion activity of increasingconcentrations of cis-2-decenoic acid against biofilm cultures of P.aeruginosa grown in microtiter plates. These results demonstrated thatthe cis isomer (cis-2-decenoic acid) was active over a concentrationrange of 1.0 nanomolar to 10 millimolar, showing greater dispersionactivity at 1.0 nanomolar than un-concentrated spent culture medium(i.e.: higher than the naturally occurring inducer). Microscopy revealedthat the activity of cis-2-decenoic acid as a dispersion inducer wassimilar to CSM activity, completely disrupting a biofilm microcolony asshown in FIG. 13. The activity of cis-2-decenoic acid was also testedagainst E. coli, S. pneumonia, P. mirabilis, S. pyogenes, B. subtilis,S. aureus, and C. albicans biofilm cultures, resulting in similarresults to those obtained for CSM (FIG. 11).

This study has shown that a small messenger fatty acid molecule,cis-2-decenoic acid, is produced by P. aeruginosa in batch and biofilmculture. This molecule has been demonstrated to induce a dispersionresponse in biofilms formed by P. aeruginosa and a range ofgram-negative and gram-positive bacteria and in yeast. The dispersionresponse is a mechanism to escape starvation conditions within apopulation, allowing fixed cells the opportunity to migrate to a morefavorable environment and thin out the population that remains, allowingcells to obtain increased nutrients. When biofilm microcolonies aresmall, the inducer, which accumulates in the extracellular matrix isremoved by diffusive and advective transport. This removal is notpossible in batch systems. When cell clusters attain a dimension wherethe inducer is not adequately washed out from the interior (the rate ofdiffusion being exceeded by the rate of production), the inducer is ableto attain a concentration necessary for activation of the dispersionresponse, releasing cells from the biofilm. The discovery of acell-to-cell signaling molecule responsible for biofilm dispersionallows the exogenous induction of the transition of biofilm bacteria toa planktonic state. Use of this dispersion inducer is likely to resultin enhanced treatment options in combating biofilm-related infectionsand in the control of microbial biofouling.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed:
 1. A method of treating or preventing a conditioncaused by a biofilm in a subject, said method comprising: providing asubject having, or susceptible to, a condition mediated by a biofilmproduced by a microorganism, whereby the biofilm comprises a matrix andthe microorganism on a surface and administering to the subject adispersion inducer comprising cis-2-decenoic acid under conditionseffective for the condition caused by a biofilm in the subject to betreated or prevented.
 2. The method of claim 1 further comprising:administering to the subject, in conjunction with said administering thedispersion inducer, an antimicrobial treatment selected from the groupconsisting of one or more of biocides, surfactants, antibiotics,antiseptics, detergents, chelating agents, virulence factor inhibitors,ultrasonic treatment, radiation treatment, thermal treatment, andmechanical treatment.
 3. The method of claim 1, wherein a subject withburns is treated.
 4. The method of claim 1, wherein a subject withdental plaque, dental caries, gingival disease, and/or oral infection istreated.
 5. The method of claim 4, wherein said administering is carriedout with a dentifrice, mouthwash, dental floss, gum, strip, or brush. 6.The method of claim 1, wherein a subject with acne or otherbiofilm-associated skin infections on the skin is treated.
 7. The methodof claim 1, wherein a subject with a chronic biofilm-associated diseaseis treated.
 8. The method of claim 7, wherein the chronicbiofilm-associated disease is selected from the group consisting ofmiddle ear infection, osteomyelitis, prostatitis, cystic fibrosis,colitis, vaginitis, urethritis, and gastric or duodenal ulcer.
 9. Themethod of claim 1, wherein said dispersion inducer is administered at aconcentration of 0.01 μM to 30 mM.
 10. The method of claim 1, whereinsaid dispersion inducer is administered as a composition having a pH of1.5 to 4.9.
 11. The method of claim 1, wherein said dispersion induceris administered as a composition having a pH of 4.5 to 8.0.
 12. Themethod of claim 11, wherein said composition has a pH of 6.8 to 7.4. 13.The method of claim 1, wherein said dispersion inducer is administeredas a composition having a pH of 8.0 to 9.8.
 14. The method of claim 1,wherein said dispersion inducer is administered as a compositionformulated so that said dispersion inducer is non-bacteriocidal.
 15. Themethod of claim 1, wherein said dispersion inducer is administered at aconcentration of less than 0.5 percent by weight.
 16. A method oftreating or inhibiting formation of a biofilm on a surface, said methodcomprising: providing a surface having or being susceptible to formationof a biofilm produced by a microorganism, whereby the biofilm comprisesa matrix and the microorganism on the surface and administering to thesurface a dispersion inducer comprising cis-2-decenoic acid underconditions effective for formation of the biofilm on the surface to betreated or inhibited.
 17. The method of claim 16, wherein the surface isa contact lens.
 18. The method of claim 16, wherein the surface is anindwelling medical device selected from the group consisting ofcatheters, respirators, and ventilators.
 19. The method of claim 16,wherein the surface is an implanted medical device selected from thegroup consisting of stents, artificial valves, joints, sutures, staples,pacemakers, bone implants, and pins.
 20. The method of claim 16, whereinthe surface is selected from the group consisting of drains, tubs,kitchen appliances, countertops, shower curtains, grout, toilets,industrial food and beverage production facilities, flooring, and foodprocessing equipment.
 21. The method of claim 16, wherein the surface isa heat exchanger surface or a filter surface.
 22. The method of claim16, wherein the surface is a marine structure selected from the groupconsisting of boats, piers, oil platforms, water intake ports, sieves,and viewing ports.
 23. The method of claim 16, wherein the surface isassociated with a system for water treatment and/or distribution. 24.The method of claim 22, wherein the system for water treatment and/ordistribution is selected from the group consisting of a system fordrinking water treatment and/or distribution, a system for pool and spawater treatment, a system for treatment and/or distribution of water inmanufacturing operations, and a system for dental water treatment and/ordistribution.
 25. The method of claim 16, wherein the surface isassociated with a system for petroleum drilling, storage, separation,refining, distribution, and/or porous medium from which the petroleum isextracted.
 26. The method of claim 24, wherein the system for petroleumdrilling, storage, separation, and/or distribution is selected from thegroup consisting of a petroleum separation train, a petroleum container,petroleum distributing pipes, and petroleum drilling equipment.
 27. Themethod of claim 16 further comprising: administering to the surface, inconjunction with said administering the dispersion inducer, at least oneantimicrobial treatment selected from the group consisting of biocides,surfactants, antibiotics, antiseptics, detergents, chelating agents,virulence factor inhibitors, ultrasonic treatment, radiation treatment,thermal treatment, and mechanical treatment.
 28. The method of claim 27,wherein the dispersion inducer and the antimicrobial treatment areadministered simultaneously.
 29. The method of claim 27, wherein thedispersion inducer and antimicrobial treatment are administeredseparately.
 30. The method of claim 27, wherein the dispersion induceris impregnated in the surface.
 31. The method of claim 27, wherein thedispersion inducer is administered in a copolymer or a gel coating overthe surface.
 32. The method of claim 27, wherein said dispersion induceris administered in a concentration of 0.01 μM to 30 mM.
 33. The methodof claim 27, wherein said dispersion inducer is administered as acomposition having a pH of 1.5 to 4.9.
 34. The method of claim 27,wherein said dispersion inducer is administered as a composition havinga pH of 4.5 to 8.0.
 35. The method of claim 34, wherein said compositionhas a pH of 6.8 to 7.4.
 36. The method of claim 27, wherein saiddispersion inducer is administered as a composition having a pH of 8.0to 9.8.
 37. The method of claim 16, wherein said dispersion inducer isadministered as a composition formulated so that said dispersion induceris non-bacteriocidal.
 38. The method of claim 16, wherein saiddispersion inducer is administered at a concentration of less than 0.5percent by weight.
 39. The method of claim 16, wherein the surface is abone implant.
 40. A method comprising: a) providing contact lenses and asolution comprising a dispersion inducer at a concentration less than0.5% by weight, said inducer comprises cis-2-decenoic acid and b)treating said contact lenses with said solution.
 41. The method of claim40, wherein said solution is substantially ethanol-free.
 42. The methodof claim 40, wherein said solution is substantially formaldehyde-free.43. The method of claim 40, wherein said pH of said solution issubstantially neutral.
 44. The method of claim 40, wherein said contactlenses are stored in said solution.
 45. The method of claim 40, whereinsaid inducer is formulated in a non-salt form.
 46. The method of claim40, wherein said solution further comprises at least one componentselected from the group consisting of biocides, surfactants,antibiotics, antiseptics, detergents, chelating agents, and virulencefactor inhibitors.
 47. A method comprising: a) providing a subject witha skin condition and a solution having a pH greater than 5, saidsolution comprising a dispersion inducer at a concentration less than0.5% by weight, said inducer comprises cis-2-decenoic acid and b)treating said skin condition with said solution.
 48. The method of claim47, wherein said solution is substantially ethanol-free.
 49. The methodof claim 47, wherein said solution is substantially formaldehyde-free.50. The method of claim 47, wherein said pH of said solution issubstantially neutral.
 51. The method of claim 47, wherein said solutionis formulated as a cream.
 52. The method of claim 47, wherein saidinducer is formulated in a non-salt form.
 53. The method of claim 47,wherein said solution further comprises at least one component selectedfrom the group consisting of biocides, surfactants, antibiotics,antiseptics, detergents, chelating agents, and virulence factorinhibitors.